Human respiratory syncytial virus (RSV) is the most important viral agent of pediatric respiratory tract disease worldwide and also is important in adults in general and in the elderly and bone marrow transplant recipients in particular. Obstacles to vaccine development include the poor growth of the virus in cell culture, the semi-permissive nature of infection in most animal models, the difficulty of achieving an appropriate balance between immunogenicity and attenuation, and the inefficiency of the immune response in the very young infant. We previously developed a method for producing infectious RSV entirely from cDNA clones, whereby defined changes can be introduced into infectious virus via the cDNA intermediate. This is being used on an ongoing basis to construct various recombinant vaccine candidates that are attenuated by the introduction of various combinations of (1) point mutations identified in biologically-derived strains that had been attenuated by chemical mutagenesis, and (2) deletion of the nonessential NS1, NS2, SH, G and M2-2 accessory genes. We also are constructing and evaluating chimeras between human RSV and bovine RSV, the latter being a related virus that exhibits a strong host range restriction in primates and thus is naturally attenuated. These chimeras will provide new vaccine candidates and also will address the basis for the host range restriction of bovine RSV. In an initial bovine/human RSV construct, substitution of the G and F protective antigen genes of human RSV into the bovine RSV backbone resulted in a virus that replicated more efficiently than bovine RSV in chimpanzees but nonetheless remained over attenuated. The construction of new chimeras containing further additions of human RSV genes is expected to identify viruses with an appropriate level of attenuation. We also are investigating strategies for engineering a live virus vaccine to increase its immunogenicity. In one strategy, we moved the G and F genes, encoding the major neutralization and protective antigens, from their natural positions downstream in the gene order to positions immediately following the major viral promoter. This resulted in a 3- to 4-fold increase in the expression of each protein and provided a modest increase in immunogenicity. As another strategy, we engineered recombinant RSV to express various cytokines and chemokines in order to enhance immunogenicity and, in some cases, attenuate viral infection. As one example, RSV was engineered to express murine granulocyte macrophage colony-stimulating factor (GM-CSF). When assayed by intranasal infection of mice (in parallel with a control virus expressing an irrelevant insert), the RSV-mGM-CSF recombinant exhibited a substantial attenuation of virus replication but nonetheless stimulated a very substantial increase in the accumulation and activation of pulmonary dendritic cells and macrophages compared to the matched control. This provides a basis for increased antigen presentation. This strategy was explored further in rhesus monkeys in a study that used a parainfluenza type 3 virus (PIV3) rather than RSV because the former virus replicates more efficiently in monkeys and would provide a more realistic appraisal. The PIV3 that was used was a recombinant chimeric virus called rB/HPIV3 that consists of a bovine PIV3 backbone bearing the F and HN protective antigen genes from human PIV3. This virus is attenuated due to a natural host range restriction conferred by the bovine PIV3 backbone and is a promising candidate to be a HPIV3 vaccine. When engineered to express human GM-CSF, this recombinant virus induced three- to six-fold higher levels of PIV3-specific serum antibodies compared to a matched control, and also induced several-fold more interferon-gamma-secreting T lymphocytes in the peripheral blood, a measure of stimulation of cellular immunity. Thus, it is possible to substantially increase the immunogenicity of an attenuated vaccine virus. We also evaluated the strategy of using rB/HPIV3 as a vector to express the major protective antigens, the G and F proteins, of RSV antigenic subgroups A and B. This strategy would obviate the use of infectious RSV and the problems of its poor growth and physical instability. The G and F genes of subgroups A and B were introduced singly or as subgroup-matched pairs into the promoter-proximal position of the B/HPIV3 vector. Evaluation in rodents and rhesus monkeys showed that the rB/HPIV3/RSV viruses were marginally more attenuated than the rB/HPIV3 parent, presumably due to the added genes, but were equivalent to rB/HPIV3 and RSV in immunogenicity against HPIV3 and RSV, respectively. The use of PIV3 as a vector for RSV antigens provides an approach that overcomes difficulties inherent in working with infectious RSV and provides a combined vaccine against HPIV3 and both RSV antigenic subgroups, representing the three most imporant viral agents of pediatric respiratory tract disease worldwide for which there is a pressing, unmet need for vaccines.